Requirement of multiple SH3 domains of Nck for ligand binding

Livius Wunderlich, Ágnes Gohér, Anna Faragó, Julian Downward, László Buday

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

The Nck adaptor protein comprises a single C-terminal SH2 domain and three SH3 domains. The domain structure of Nck suggests that Nck links tyrosine kinase substrates to proteins containing proline-rich motifs. Here we show that Bcr/Abl tyrosine kinase, and three tyrosine phosphorylated proteins (115, 120 and 155 kDa) are co-immunoprecipitated with antibody against Nck from lysates of the human leukaemia cell line K562. By means of affinity purification with the Nck-binding phosphopeptide EPGPY(P)AQPSV, we could also detect the association of endogenous Nck with the proto-oncogene product Cbl. An investigation of the nature of interactions revealed that Bcr/Abl, Cbl, and the 155-kDa tyrosine phosphotyrosine bind exclusively to the SH3 domains of Nck. In addition, none of the single SH3 domains of Nck expressed as glutathione-S-transferase (GST) fusion proteins is able to interact with the proline-rich ligands. However, combined first and second SH3 domains have the capacity to bind Bcr/Abl, Cbl and p155. Mutations of conserved tryptophan to lysine in either of the combined first and second SH3 domains completely abolish ligand binding. These data suggest that cooperation exists among the SH3 domains of Nck for a high-affinity binding of proteins containing proline-rich motifs. Copyright (C) 1999 Elsevier Science Inc. All rights reserved.

Original languageEnglish
Pages (from-to)253-262
Number of pages10
JournalCellular Signalling
Volume11
Issue number4
DOIs
Publication statusPublished - Apr 1 1999

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Keywords

  • K562 cells
  • Nck, Bcr/Abl, Cbl, SH2/SH3 domains

ASJC Scopus subject areas

  • Cell Biology

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